To begin, obtain the retinal strips from mice and wash them five times in 0.1 molar sodium cacodylate buffer for 15 minutes. Then incubate the strips with potassium ferrocyanide and osmium tetroxide solution in the dark on ice for 1.5 hours. Wash the strips three times in double distilled water and incubate them with 1%thio-carbo-hydrazide.
After rinsing with double distilled water, sequentially treat the retina strips with osmium tetroxide, uranyl acetate, and lead nitrate. Dehydrate the retina strips with increasing concentrations of ethanol. Then, treat retina strips with a solution containing equal ethanol and acetone for 20 minutes, followed by two acetone washes for 20 minutes each.
Sequentially incubate the retina strips in acetone resin mixtures in the dark at room temperature. Infiltrate the retina strips with pure resin for 24 hours at 45 degrees Celsius, followed by fresh resin for 48 hours at 60 degrees Celsius. The focused ion beam scanning electron microscopy of retinal photoreceptor terminals using this method preserves cell membrane structure and vesicle outlines more clearly than the traditional double fixation method.
